In studies of hypoxic injury to freshly isolated proximal tubules during the current grant period, we recognized a novel, large magnitude, metabolism-independent, cytoprotective action of glycine and a limited number of structurally-related small neutral amino acids. We also extended our prior observation that moderate acidosis is protective for proximal tubule cells. We hypothesize that availability of glycine in combination with alterations of intracellular pH plays a major role, possibly the deciding one, in determining acute tubule cell alterations of intracellular pH plays a major role, possibly the deciding one, in determining acute tubule cell viability during oxygen deprivation- reoxygenation injury, including the tubule cell damage which contributes to ischemic acute renal failure in vivo. Glycine is the most important of the two factors and can potentially impact on a variety of other injury settings and cell types as well. During the next funding period, using both freshly isolated proximal tubules and cultured tubule epithelial cells, we plan studies to further understand the injury conditions under which glycine is protective, to determine what factors ultimately limit glycine cytoprotection, and to elucidate the specific cellular target(s) of glycine. To these ends we have five Specific Aims: 1) Using improved methods for assessing cytosolic free calcium (Caf) in the low micromolar range that may be most relevant to injury states, we plan to more precisely determine the Caf requirements for induction of the phospholipid hydrolysis and apical membrane remodeling that occurs despite glycine cytoprotection and to assess whether these processes are further modulated by low pH conditions. Additionally, we will employ confocal microscopy and biochemical techniques to study the contribution of cytoskeletal alterations to this membrane remodeling and their role in glycine cytoprotection. 2) We will extend our analysis of oxygen deprivation and related forms of ATP depletion-induced injury in isolated tubules to analyze the mechanisms for lethal membrane damage that occurs despite high glycine and low pH conditions. 3) We will assess the mechanisms of a highly glycine-sensitive form of reoxygenation injury to the isolated proximal tubules. 4) We have found that glycine can be protective against oxidant injury to both freshly isolated and cultured tubule cells, but protection is more selective than for other forms of injury and may not be expressed against Fe-mediated damage to the fresh proximal tubule. We will determine the applicability of glycine cytoprotection to oxidant-induced tubule injury. 5) We have demonstrated strong expression of glycine cytoprotection during injury to MDCK cells and have been able to clone differentially sensitive lines. We will characterize the basis for clonal variation in sensitivity, attempt to isolate additional lines with even more desirable properties, and, using these lines, we will expand our structure activity studies of protective compounds and initiate work to delineate whether specific cytoskeletal proteins are the primary targets for glycine. Our overall aim remains to understand the cellular pathophysiology of acute renal failure in the expectation that this will lead to improved methods of prevention and treatment. Glycine cytoprotection is an extraordinarily powerful tool for this purpose with implications for injury to the kidney and other organs